Microfabricated capillary array electrophoresis device and method

ABSTRACT

A capillary array electrophoresis (CAE) micro-plate with an array of separation channels connected to an array of sample reservoirs on the plate. The sample reservoirs are organized into one or more sample injectors. One or more waste reservoirs are provided to collect wastes from reservoirs in each of the sample injectors. Additionally, a cathode reservoir is also multiplexed with one or more separation channels. To complete the electrical path, an anode reservoir which is common to some or all separation channels is also provided on the micro-plate. Moreover, the channel layout keeps the distance from the anode to each of the cathodes approximately constant.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under Grant No.DE-FG-91ER61125, awarded by the U.S. Department of Energy, and Grant No.HG01399, awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

This invention relates to electrophoresis generally, and moreparticularly, to an apparatus and method for performing capillary arrayelectrophoresis on microfabricated structures.

In many diagnostic and gene identification procedures such as genemapping, gene sequencing and disease diagnosis, deoxyribonucleic acid(DNA), ribonucleic acid (RNA) or proteins are separated according totheir physical and chemical properties. In addition to DNA, RNA orproteins, other small molecule analytes may also need to be separated.

One electrochemical separation process is known as electrophoresis. Inthis process, molecules are transported in a capillary or a channelwhich is connected to a buffer-filled reservoir. An electric field inthe range of kilovolts is applied across both ends of the channel tocause the molecules to migrate. Samples are typically introduced at ahigh potential end and, under the influence of the electric field, movetoward a low potential end of the channel. After migrating through thechannel, the separated samples are detected by a suitable detector.

Typically, electrophoretic separation of nucleic acids and proteins iscarried out in a gel separation medium. Although slab gels have played amajor role in electrophoresis, difficulties exist in preparing uniformgels over a large area, in maintaining reproducibility of the differentgels, in loading sample wells, in uniformly cooling the gels, in usinglarge amounts of media, buffers, and samples, and in requiring long runtimes for extended reading of nucleotides. Moreover, slab gels are notreadily amenable to a high degree of multiplexing and automation.Recently, micro-fabricated capillary electrophoresis (CE) devices havebeen used to separate fluorescent dyes and fluorescently labeled aminoacids. Additionally, DNA restriction fragments, polymerase chainreaction (PCR) products, short oligonucleotides and even DNA sequencingfragments have been effectively separated with CE devices. Also,integrated micro-devices have been developed that can perform polymerasechain reaction amplification immediately followed by amplicon sizing,DNA restriction/digestion and subsequent size-based separation, andcells sorting and membrane lysis of selected cells. However, thesemicro-fabricated devices only perform analysis on one channel at a time.For applications such as population screening or DNA sequencing, such asingle channel observation and analysis results in an unacceptable delayfor screening many members of a population.

SUMMARY OF THE INVENTION

The invention provides a capillary array electrophoresis (CAE)micro-plate. The micro-plate has an array of separation channelsconnected to an array of sample reservoirs on the plate. The samplereservoirs are organized into one or more sample injectors. A wastereservoir is provided to collect wastes from sample reservoirs in eachof the sample injectors. Additionally, a cathode reservoir ismultiplexed with one or more separation channels. An anode reservoirwhich is common to some or all separation channels is also provided onthe micro-plate. Moreover, the distance from the anode to each of thecathodes is kept constant by deploying folded channels. The corners onthese turns may be right angle turns or more preferably, smooth curvesto improve electrophoretic resolution.

In one aspect, the reservoir layout on the substrate separates thesample reservoirs by a predetermined spacing to facilitate thesimultaneous loading of multiple samples.

In another aspect, cathode, anode and injection waste reservoirs arecombined to reduce the number of holes N in the substrate to about 5/4Nwhere N is the number of samples analyzed.

In another aspect, the separation channels are formed from linearsegments.

In another aspect, the separation channels are formed from curvilinearsegments, which may include radial segments.

In yet another aspect, the separation channels span from the perimeterof the plate to the central region of the plate. The separation channelsmay span the plate in a linear or a radial fashion.

In yet another aspect, a CAE micro-plate assembly is formed using amicro-plate, a reservoir array layer, and an electrode array. Theassembly simplifies sample handling, electrode introduction and allowsan increased volume of buffer to be present in the cathode and anodereservoirs.

Advantages of the invention include the following. The micro-plate ofthe present invention permits analysis of a large number of samples tobe performed at once on a small device. Moreover, the micro-plate allowssamples to be easily loaded while minimizing the risk of contamination.Additionally, the micro-plate is easy to electrically address. Further,the micro-plate supports a wide variety of formats that can providehigher resolution separation and detection of samples, faster separationand detection of samples, or separation and detection of more samples.

Other features and advantages will be apparent from the followingdescription and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of the specification, schematically illustrate the presentinvention and, together with the general description given above and thedetailed description given below, serve to explain the principles of theinvention.

FIG. 1 is a capillary array electrophoresis (CAE) micro-plate layout.

FIG. 2 is a schematic illustration of the sample injector of FIG. 1.

FIGS. 3A-3D are illustrations of the operation of the sample injector ofFIG. 2.

FIG. 4A is an exploded perspective view of a CAE micro-plate assembly.

FIG. 4B is a cross-sectional side view of the CAE micro-plate assemblyof FIG. 4A.

FIG. 5 is an illustration of a laser excited galvo-scanner inconjunction with a CAE micro-plate.

FIGS. 6A and 6B are images of separations of genetic markers forhereditary hemochromatosis.

FIG. 7 is a plot of electropherograms generated from the images of FIGS.6A and 6B.

FIG. 8 is a second CAE micro-plate layout.

FIG. 9 is a third CAE micro-plate layout.

FIG. 10 is a schematic illustration of a sample injector of FIG. 9.

FIG. 11 is an enlarged view of a perimeter portion of the CAEmicro-plate layout of FIG. 9.

FIG. 12 is an enlarged view of a center portion of the CAE micro-platelayout of FIG. 9.

DESCRIPTION

Referring now to FIG. 1, a capillary array electrophoresis (CAE)micro-plate 10 is shown. The micro-plate 10 has an array of capillariesor separation channels 50 etched thereon. In one embodiment of FIG. 1,48 individual separation channels are etched in a 150 micron (μm)periodic array. In this embodiment, the separation channels 50 branchout to an 8×12 array of sample reservoirs 101, each of which is spaced apredetermined distance apart to facilitate loading with an 8-tippedpipetter. In this case, each sample reservoir 101 is spaced in onedimension nine millimeters apart from another sample reservoir. Theseparation channels 50 extend by a first predetermined distance from aninjection region to an anode reservoir 180 and by a second predetermineddistance from an injector group 100 to a cathode reservoir 120. Thefirst predetermined distance may be about 10 centimeters, while thesecond predetermined distance may be about 1.8 centimeters.

Each of the sample reservoirs 101 belongs to an injector group such asone of injector groups 100-116. Additionally, injector groups 100, 102and 104 are connected to a cathode reservoir 120. Although the cathodereservoir 120 is connected to three sample injectors 100, 102 and 104,other cathode injectors may be connected to more than three sampleinjectors. For instance, a cathode injector 130 is connected to sampleinjectors 106, 108, 110, 112, 114 and 116.

The anode reservoir 180 is placed in a non-symmetrical manner in thiscase to avoid a conflict with a scanning system. Moreover, the distancefor paths from the anode reservoir 180 to any one of cathodes 120 or 130is identical for all separation channels. The equal distance is achievedby providing folded paths connecting certain sample reservoirs that areclose to the anode 180 to increase the path length and to achieve auniform distance between the anode reservoir 180 and the cathodereservoirs 120 and 130 for all sample reservoirs.

In the embodiment of FIG. 1, the number of holes H in the micro-plate 10is about 5N/4, and more exactly, 5N/4+7, where N is a number of samples.As the embodiment of FIG. 1 addresses 96 samples in parallel, 127 holesare required to be drilled. This number of holes is close to atheoretical minimum number of holes of N+3. The reduction in hole countsis advantageous as fewer holes need to be drilled into the micro-plate10, thereby increasing manufacturing efficiency as well as decreasingthe potential for defects in the production of micro-plates, as causedby mechanical stress associated with the drilling process. Anotherreason for multiplexing the cathode, anode and waste reservoirs is tomake it more feasible to fit 96 separation system on a single substrate.The above advantages are also applicable in the event that the holes areformed by a molding process or a bonding process in lieu of the drillingprocess.

Turning now to FIG. 2, details of the sample injector 100 of FIG. 1 areshown. The sample injector 100 has a plurality of sample reservoirs 200,204, 220 and 224. Sample reservoirs 200 and 220 contain a first sample,while sample reservoirs 204 and 224 contain a second sample.

The sample injector 100 also has a first separation channel 202 and asecond separation channel 222. The sample injector 100 thus permits aserial analysis of two different samples on each separation channel. Thefirst and second separation channels 202 and 222 are connected to awaste reservoir 208 by a cross channel 207. The sample injector 100 alsohas a cathode end 210 as well as an anode end 212. The cathode and anodeends 210 and 212 are at opposite ends of the first separation channel202. Similarly, a second cathode end 214 is connected to a second anodeend 216 by a separation channel 222 that is connected to the wastereservoir 208. As illustrated below, by a proper biasing of the anodereservoirs 211 and 212, cathode reservoirs 200 and 214, samplereservoirs 200, 204, 220, 224, and waste reservoir 208, samples may bemoved from their respective sample reservoirs 200, 204, 220 and 224through the cross channel to the waste reservoir thereby facilitating aninsertion into the separation channel.

Referring now to FIGS. 3A, 3B, 3C and 3D, a process for loading a samplefrom its respective sample reservoir into the cross channel and thenperforming a separation is shown. In FIG. 3A, an injection voltage,preferably about 300 volts (3.0 V/cm), is applied between the samplereservoir 200 and the injection waste reservoir 208 to draw a samplethrough a channel that passes from the sample reservoir to the wastereservoir and crosses the separation channel.

In FIG. 3B, a separation voltage of about 3700 volts (300 V/cm), forexample, is applied between the cathode end 210 and the anode end 212.This causes the electrophoretic separation of the sample. In addition, aback-bias of the potential between the sample reservoir 200 and theinjection waste reservoir 208 is applied. Preferably, the back biasingvoltage is about 720 volts. The back-biasing operation clears excesssamples from the injection cross-channel 213. As illustrated in FIG. 3B,a 100 μm sample plug is injected and any residual sample is pulled awayfrom the injection region to avoid tailing side-effects.

FIGS. 3C and 3D represents analogous injections from the second samplereservoir 204. Although the embodiment of FIGS. 2 and 3A-3D operates ontwo samples, four samples may be injected onto a single capillarywithout any significant cross-contamination.

The process of etching patterns into a representative micro-plate isdiscussed next. In one microfabricated embodiment, Borofloat glasswafers available from Schott Corporation of Yonkers, N.Y. are pre-etchedin 49% HF for 15 sec and cleaned before deposition of an amorphoussilicon sacrificial layer of about 1500 Å in a plasma enhanced chemicalvapor deposition (PECVD) system. The wafers are primed withhexamethyldisilazane, spin coated at 5000 rpm with a photoresist such asa 1818 photoresist available from Shipley Corp. of Marlborough, Mass.The photoresist is developed in a 1:1 mixture of Microposit developerconcentrate available from Shipley and water. The wafers are thensoft-baked at 90° C. for 30 minutes. The mask pattern is transferred tothe substrate by exposing the photoresist to ultraviolet radiation in aQuintel contact mask aligner. The mask pattern is transferred to theamorphous silicon by a CF₄ plasma etch performed in the PECVD reactor.The wafers are etched in a 49% HF solution for about 3 minutes at anetch rate of 7 μm/min to form a final etch depth of 21 μm and channelwidth of ˜60 μm at the bonded surface. The photoresist is stripped andthe remaining amorphous silicon is removed in a CF₄ plasma etch. Holesare drilled into the etched plate using a 1.25 mm diameterdiamond-tipped drill bit, available from Crystalite Corporation ofWesterville, Ohio. The etched and drilled plate is thermally bonded to aflat wafer of similar size and type in a programmable vacuum furnace.After bonding, the channel surfaces are coated using a coating protocol.

Turning now to FIGS. 4A and 4B, an exploded view and a cross-sectionalside view of a CAE micro plate are shown. In FIG. 4A, a CAE micro-plate302 with etched separation channels 301 and a plurality of reservoirs303 formed thereon is provided. A reservoir array layer 304 is mountedabove the CAE micro-plate 302 to provide additional reservoir spaceabove the reservoirs formed on the micro-plate 302. The presence of thereservoir array layer 304 increases the volume of buffers in the cathodeand anode reservoirs and simplifies sample handling and electrodeintroduction. Preferably, the reservoir array layer 304 is a onemillimeter thick elastomer sheet which makes a watertight seal when itis in contact with the glass micro-plate 302. The reservoir array layer304 may be an elastomer such as Sylgard 184, available from Dow Corningof Midland, Mich.

The reservoir array layer 304 is placed onto the micro-plate 302 beforethe channels are filled with a separation medium. Preferably, theseparation medium is 0.75 percent weight/volume hydroxyethylcellulose(HEC) in a 1× TBE buffer with 1 μM ethidium bromide. Additionally, thereservoir array 304 fully isolates the reservoirs from each other. Theseparation channels are pressure filled with a sieving matrix from theanode reservoir 180 until all channels have been filled. The anode andcathode reservoirs 180 and 120 are then filled with a 10× TBE buffer toreduce ion depletion during electrophoresis. The sample reservoirs arerinsed with deionized water. Samples are then loaded from a micro-titerplate using an 8-tipped pipetter.

After the reservoir array layer 304 is positioned on the micro-plate302, an electrode array 306 is placed above the reservoir array 304. Theelectrode array 306 is fabricated by placing an array of conductors suchas platinum wires through a printed circuit board. Each conductor isadapted to engage a reservoir on the micro-plate 302. Moreover, thewires are electrically connected with metal strips on the circuit boardto allow individual reservoirs of a common type to be electricallyaddressed in parallel. The electrode array 306 also reduces thepossibility of buffer evaporation. The electrode array 306 in turn isconnected to one or more computer controlled power supplies.

As shown in FIG. 4B, the reservoir array layer 304, when used inconjunction with the micro-plate 302, enlarges the effective volume ofthe reservoirs originally formed on the micro-plate 302. Moreover,electrodes from the electrode array 306 are adapted to probe thereservoirs on the micro-plate 302 and the reservoir array layer 306. Thesolutions are placed in the reservoirs by a pipetter 308.

After assembly, the CAE micro-plate 302 is probed with a galvo-scannersystem 400, as shown in FIG. 5. The system 400 measures fluorescenceusing a detector at a detection zone of the channels. During the processof electrophoresis, as a fluorescent species traverses a detection zone,it is excited by an incident laser beam. In a direct fluorescencedetection system, either the target species is fluorescent, or it istransformed into a fluorescent species by tagging it with a fluorophore.The passing of the fluorescent species across the detection zone resultsin a change, typically an increase in fluorescence that is detectable bythe system 400.

Turning now to the analysis system, the galvo-scanner 400 has afrequency-doubled YAG laser such as YAG laser available from UniphaseCorporation of San Jose, Calif. The YAG laser generates a beam which maybe a 30 mW, 532 nm beam. The beam generated by the laser 402 travelsthrough an excitation filter 404 and is redirected by a mirror 406. Fromthe mirror 406, the beam travels through a beam expander 408. Afterexpansion, the beam is directed to a dichroic beam splitter 410. Thelaser beam is directed to a galvonometer 420 which directs the beam to afinal lens assembly 422. In this manner, the beam is focused on a spotof about 5 μm where it excited flourescence from the molecules in thechannels and is scanned across the channels at 40 Hz. The resultingflourescence is gathered by the final lens and passed through thegalvomirror and the dichroic beam splitter 410 to an emission filter 412which operates in the range of about 545-620 nm. After passing throughthe emission filter 412, the beam is focused by a lens 414. Next, thebeam is directed through a pinhole 416 such as a 400 μm pinhole fordelivery to a photomultiplier (PMT) 418.

The electrode array 306 is connected to one or more power supplies 428such as a series PS300, available from Stanford Research Systems ofSunnyvale, Calif. The power supplies are connected to a computer andsoftware controlled to automatically time and switch the appropriatevoltages into the electrode array 306. The software may be written in aconventional computer language, or may be specified in a dataacquisition software such as LabVIEW, available from NationalInstruments of Austin, Tex. Data corresponding to spatially distinctfluorescent emission may then be acquired at about 77 kHz using a 16 bitA/D converter from Burr-Brown Corporation of Tucson, Ariz. Logarithmicdata compression is then applied to generate five linear orders ofdynamic measurement range. The data is obtained as a 16 bit image, andelectropherograms are then generated using a suitable software such asIPLab, available from Signal Analytics, Vienna, Va., to sum data pointsacross each channel. A detection of all lanes with a 0.09 secondtemporal resolution has been achieved by the system 400.

EXPERIMENTS

An electrophoretic separation and fluorescence detection of HFE, amarker gene for hereditary hemochromatosis, was performed to demonstratethe high-throughput analysis of biologically relevant samples using theCAE micro-plates of the present invention. HFE is a genetic disorderthat causes a buildup of iron in tissues resulting over time in disease.The buildup primarily affects the liver. Between 0.1 and 0.5% of theCaucasian population are homozygous for an HFE C282Y variant responsiblefor this disease. If detected early, treatment can be initiated and longterm effects avoided. To screen the population for this marker gene, ahigh throughput screening system is needed.

In this experiment, samples were prepared using PCR amplification anddigestion to assay the C282Y mutation in the HFE gene. This G A mutationat nucleotide 845 creates a Rsa I restriction site in the HFE gene. DNAmaterials were isolated from peripheral blood leukocytes using standardmethods. A segment of an HFE exon containing the variant site wasamplified with the following primers:

HH-E4B: 5'GACCTCTTCAGTGACCACTC3'

HC282R: 5'CTCAGGCACTCCTCTCAACC3'

The HC282R primer is a primer discussed in Feder et al., Nature Genet.13, 399-408 (1996), hereby incorporated by reference. The HH-E4B primercontains a 5' biotin tag. The 25 μl amplification reaction contained 10mM Tris-HCl (pH=8.8), 50 mM KCl, 0.75 mM MgCl₂, 0.2 mM dNTPs, 7.5 pM ofeach primer and 1.5 U AmpliTaq DNA, available from Perkin Elmer,Branchburg, N.J. The PCR was carried out under three consecutiveconditions: 5 cycles (95° C. for 1 min, 64° C. for 1 min, 72° C. for 1min), 5 cycles (95° C. for 1 min, 60° C. for 1 min, 72° C. for 1 min),and 25 cycles (95° C. for 1 min, 56° C. for 1 min, 72° C. for 1 min).The restriction digestion of amplified product was carried out by adding4 μl of each amplified sample to 6 μl buffer containing 2 U Rsa I(Sigma, St. Louis, Mo.) and digesting for 90 minutes at 37° C. Sampleswere dialyzed against DI water on a 96 sample dialysis plate, availablefrom Millipore, Bedford, Mass. Sample types were initially establishedby separation of restriction fragments on 1% Agarose-3% SeaPlaque gel,available from FMC Bioproducts, Rockland, Me, in 0.5×TBE. Gels werestained in 0.5 μg/ml ethidium bromide for 30 minutes and visualized on aUV transilluminator, a Spectroline model TR-302, using a 123-bp ladder,available from Life Technologies Inc., Gaithersburg, Md., to determinefragment sizes.

FIG. 6A and 6B present images of separations of 96 HFE samples on a CAEmicro-plate. The 96 samples were separated in two runs of 48 samples,corresponding to two injection reservoirs per channel. In thisexperiment, nineteen different samples were dispersed among the 96sample wells, giving a 5-fold redundancy in sample analysis. An originalimage 500 was obtained for the first injection, while an original image504 was obtained for the second injection. Additionally, expanded images502 and 506, corresponding to original images 500 and 504 are shown. Thewidth of the electrophoretic image shown is 7.4 mm for 48 lanes and thecomplete analysis of 96 samples was performed in under 8 minutes. Theexpanded images show that the bands are of high intensity andresolution. The image exhibits a smile with the right lanes about 20seconds faster than the left. This is caused by a gradient in theelectrophoresis voltages caused by the placement of the anode to theside of the injection region to ensure adequate clearance from thescanning lens.

FIG. 7 presents the 96 electropherograms obtained from the images inFIGS. 6A and 6B. All electropherograms have been shifted to align a167-bp doublet in order to compare the separations. The 167-bp fragmentappears as a doublet due to a partial biotinylation of the HH-E4Bprimer, as the biotinylated form accounts for the slower migratingfragment in the doublet. The 167-bp doublet provides a useful referencepoint for the alignment of electropherograms to compare separations andallows an accurate genotyping without requiring a sizing ladder. Asshown in FIG. 7, an average distance between the 111 and 140-bp bands is7.3 seconds with a standard deviation (SD) of 0.8 second and 0.6 second,respectively, for the first injection and 6.6 sec with a SD of 1.1second and 0.5 second, respectively, for the second injection. Using at-test, the typings for both injections are determined to be at about a99.9% confidence level.

Referring to FIG. 8, a second embodiment of the CAE micro-plate 600 isshown. In FIG. 8, the micro-plate 600 is an array of injectors, each ofwhich includes waste reservoirs 602 and 608, sample reservoirs 604, 606,610 and 612. Each injector unit is connected to one of two cathodereservoirs 614 or 616, respectively. Additionally, each injector unit isconnected to one capillary in an array of capillaries or channels 620.The capillaries or channels 620 are connected to an anode 630. In thisdesign, 96 samples can be analyzed by injecting four samples serially ona single capillary. Further, 24 separation capillaries or channels areused to analyze the material in 96 sample reservoirs. Moreover, each ofthe injector units has two waste reservoirs. In total, the embodiment ofFIG. 8 has a hole count of 3N/2+3.

Referring now to FIG. 9, a third embodiment of the CAE micro-plate 650is disclosed. In the CAE micro-plate 650 of FIG. 9, cathode reservoirs652 are positioned on a perimeter of the CAE micro-plate 650.Additionally, an anode reservoir 660 is positioned in the center of theCAE micro-plate 650. Separation channels or capillaries may emanate froman outer perimeter of the micro-plate 650 toward the center of themicro-plate 650 in a spiral pattern if longer separation channels aredesired. Alternatively, if short paths are desired, the separationchannels or capillaries may simply be a straight line connecting theperimeter of the micro-plate 650 to the center 660 of the CAEmicro-plate 650.

Turning now to FIGS. 10 and 11, an injector unit of the CAE micro-plateof FIG. 9 and its position on a perimeter of the micro-plate of FIG. 9are illustrated in detail. In FIG. 10, two separation channels orcapillaries 670 and 671 are connected to a common waste reservoir 672and a common cathode reservoir 674. Additionally, the separationchannels 670 and 671 are connected to sample reservoirs 676 and 678. Asshown in FIGS. 10 and 11, the connections between the sample and wastereservoirs may intersect in an off-set manner.

Referring now to FIG. 12, the common anode 660 of FIG. 9 is illustratedin detail. As shown in FIG. 11, a plurality of separation channels orcapillaries 800-810 form a curvilinear pattern, which may be a radialpattern, converging on a central region 820. From the central region820, the separation channels or capillaries form a passageway from theperimeter of the central region 820 to the common anode reservoir 660 atthe center of the CAE micro-plate. The center area 820 is the area wherea rotating scanner may be used for detection purposes.

Samples may be loaded manually or automatically. Serial injections maybe used to increase the sample throughput with a predetermined number ofcapillaries. Moreover, while one embodiment of the present inventioninjects two samples per channel, an injection of four samples perchannel may be used to analyze 192 samples per plate. Further, anincrease in the number of capillaries on the CAE micro-plate wouldincrease the throughput correspondingly without introducing any samplecontamination. Moreover, the plate may be made of glass or plastic.

In addition, the scanning detection system may be altered by invertingits objective lens and scanning from below. Placing of the optics belowthe plate would permit facile manipulation and introduction of samples.The inverted scanning would also avoid spatial conflict with the anodereservoir, thereby permitting a central placement of the anode.Moreover, an array of PCR reaction chambers may be used with themicro-plate of the invention to allow for integrated amplification oflow volume samples, eliminate sample handling and manual transfer, andreduce cost. Furthermore, the present invention contemplates thatelectronic heaters, thermocouples and detection systems may be used withan array of microfluidic capillaries to enhance the CAE electrophoresisprocess.

While the invention has been shown and described with reference to anembodiment thereof, those skilled in the art will understand that theabove and other changes in form and detail may be made without departingfrom the spirit and scope of the following claims.

What is claimed is:
 1. A capillary array electrophoresis plate, comprising:one or more anode reservoirs; a plurality of separation channels connected to said anode reservoirs; and one or more injectors formed in said plate, each injector including:two or more sample reservoirs coupled to said separation channels; a plurality of waste reservoirs connected to said separation channels; and at least one cathode reservoir multiplexed with two or more of said separation channels.
 2. The plate of claim 1, further comprising an electrode array coupleable to said reservoirs.
 3. The plate of claim 1, wherein the plate has an outer perimeter and a center and the separation channels connect the outer perimeter to the center.
 4. A capillary array electrophoresis plate comprising:an array of microfabricated separation channels formed at a surface of a first microfabricated substrate and a corresponding surface of a second substrate bonded to the surface of said first substrate, each of said channels including a first end and a second end; an array of sample reservoirs connected to said array of separation channels; an array of waste reservoirs connected to said array of separation channels; an array of cathode reservoirs connected to the first end of each of the separation channels; an array of anode reservoirs, wherein at least one anode reservoir is connected to the respective second ends of at least two of the separation channels; and an injector formed by an injection channel connected to a sample reservoir that crosses a separation channel and connects to a waste reservoir.
 5. A capillary array electrophoresis plate comprising:an array of microfabricated separation channels formed on a surface of a first microfabricated substrate and a corresponding surface of a second substrate bonded to the surface of said first substrate, each of said channels having a first end and a second end; an array of sample reservoirs connected to said array of separation channels; an array of waste reservoirs connected to said array of separation channels; an array of anode reservoirs connected to the second end of each of the separation channels; an array of cathode reservoirs, wherein at least one cathode reservoir is connected to the respective first ends of at least two of the separation channels; and an injector formed by an injection channel crossing one of said separation channels, said injection channel connecting one sample reservoir of said array of sample reservoirs to one waste reservoir of said array of waste reservoirs.
 6. A capillary array electrophoresis plate comprising:an array of microfabricated separation channels formed on a surface of a first microfabricated substrate and a corresponding surface of a second substrate bonded to the surface of said first substrate, each of said channels having a first end and a second end; an array of sample reservoirs connected to said array of separation channels wherein each separation channel of the array of separation channels is in fluid communication with at least one dedicated sample reservoir; and an array of waste reservoirs connected to said separation channels, wherein at least one waste reservoir is multiplexed to two or more of said channels.
 7. The capillary array electrophoresis plate of claim 4, 5, or 6, wherein both substrates are made of glass.
 8. The capillary array electrophoresis plate of claim 4, 5, or 6, wherein both substrates are made of plastic.
 9. The capillary array electrophoresis plate of claim 4 or 5, further comprising a reservoir array layer mounted above the plate, the reservoir array layer including openings positioned to couple to a combination of the sample reservoirs, the waste reservoirs, the cathode reservoirs, and the anode reservoirs.
 10. The plate of claim 9, further comprising an electrode array coupleable to said reservoir array layer.
 11. The capillary array electrophoresis plate of claim 10 wherein said electrode array is integral with the two substrates.
 12. The capillary array electrophoresis plate of claim 11, wherein the sample reservoirs are regularly spaced on the plate to receive solutions from a parallel loading device.
 13. The capillary array electrophoresis plate of claim 4 or 5, wherein the first substrate includes an array of electrodes aligned with the sample reservoirs, the waste reservoirs, the cathode reservoirs, and the anode reservoirs to make electrical contacts with a plurality of solutions in a combination of the sample reservoirs, the waste reservoirs, the cathode reservoirs, and the anode reservoirs.
 14. The capillary array electrophoresis plate of claim 4, or 5, wherein the capillary array electrophoresis plate has H holes, and wherein H is approximately equal to 5N/4, with N being the number of samples to be processed.
 15. The capillary array electrophoresis plate of claim 4, 5, or 6, wherein the capillary array electrophoresis plate is made of a combination of glass and plastic.
 16. The capillary array electrophoresis plate of claim 4, 5, or 6, further comprising an electrode array in electrical contact with the plate.
 17. The capillary array electrophoresis plate of claim 4 or 5, wherein at least one waste reservoir of said array of waste reservoirs is multiplexed with two or more of said separation channels.
 18. The capillary array electrophoresis plate of claim 6, further comprising a reservoir array layer mounted above the plate, the reservoir array layer including openings positioned to connect to a combination of the sample reservoirs and the waste reservoirs.
 19. The capillary array electrophoresis plate of claim 6, wherein the first substrate includes an array of electrodes aligned with the sample reservoirs and the waste reservoirs to make electrical contacts with a plurality of solutions in a combination of the sample reservoirs and the waste reservoirs.
 20. A capillary array electrophoresis plate, comprising:a plurality of separation channels formed in said plate; a plurality of sample reservoirs connected to said separation channels wherein each separation channel of the plurality of separation channels is in fluid communication with at least one dedicated sample reservoir; and an anode reservoir multiplexed to two or more of the plurality of separation channels.
 21. A capillary electrophoresis plate, comprising:a plurality of separation channels formed in said plate; a plurality of sample reservoirs coupled to said separation channels wherein each separation channel of the plurality of separation channels is in fluid communication with at least one dedicated sample reservoir; and a cathode reservoir multiplexed to two or more of the plurality of separation channels.
 22. A capillary array electrophoresis plate, comprising:a plurality of separation channels formed in said plate; a plurality of sample reservoirs coupled to said separation channels wherein each separation channel of the plurality of separation channels is in fluid communication with at least one dedicated sample reservoir; and a waste reservoir multiplexed to two or more of the plurality of separation channels.
 23. The plate of claim 20, 21, or 22, further comprising a reservoir array layer mounted on the plate.
 24. The plate of claim 23, further comprising an array of electrodes insertable into and positioned above said reservoir array layer.
 25. The plate of claim 20, 21, or 22, wherein said plurality of sample reservoirs are regularly spaced in said plate and adapted to engage a parallel loading device.
 26. The capillary array electrophoresis plate of claim 25, wherein the parallel loading device comprises a multi-headed pipetter.
 27. The capillary array electrophoresis plate of claim 20, 21, or 22, further comprising an electrode array in electrical contact with said plate.
 28. The capillary array electrophoresis plate of claims 1, 4, 20, 21, 22, 5 or 6, wherein,the separation channels are disposed in a radial pattern on the capillary array electrophoresis plate.
 29. A method for injecting a sample through a capillary array electrophoresis plate, comprising:connecting a cathode reservoir to respective first ends of two or more separation channels; connecting an anode reservoir to respective second ends of two or more of said separation channels; connecting a sample reservoir and a waste reservoir together by a cross channel that crosses one of said separation channels; applying an injection voltage between the sample reservoir and the waste reservoir to draw the sample into the cross channel while applying a bias voltage to the cathode reservoir and the anode reservoir to control an injection plug width; applying a running voltage between the cathode and anode reservoirs; and applying a biasing voltage to the waste reservoir and the sample reservoir to pull away residuals of the sample.
 30. A method of forming a capillary array electrophoresis plate, comprising:forming a plurality of separation channels in said plate; forming a plurality of sample reservoirs connected to said separation channels wherein each separation channel of the plurality of separation channels is in fluid communication with at least one dedicated sample reservoir; and connecting an anode reservoir to two or more of the plurality of separation channels.
 31. A method of forming a capillary array electrophoresis plate, comprising:forming a plurality of separation channels in said plate; forming a plurality of sample reservoirs connected to said separation channels wherein each separation channel of the plurality of separation channels is in fluid communication with at least one dedicated sample reservoir; and connecting a cathode reservoir to two or more of the plurality of separation channels.
 32. A method of forming a capillary array electrophoresis plate, comprising:forming a plurality of separation channels in said plate; forming a plurality of sample reservoirs connected to said separation channels wherein each separation channel of the plurality of separation channels is in fluid communication with at least one dedicated sample reservoir; and connecting a waste reservoir to two or more of the plurality of sample reservoirs.
 33. The method of claims 29, 30, 31 or 32, wherein,the separation channels are disposed in a radial pattern on the capillary array electrophoresis plate.
 34. A method of forming a capillary array electrophoresis plate, comprising:forming an array of microfabricated separation channels; forming an array of sample reservoirs; connecting the array of sample reservoirs to the array of microfabricated separation channels; connecting one or more cathode reservoirs to said array of separation channels, wherein at least one cathode reservoir is connected to two or more separation channels of said array of separation channels; connecting one or more anode reservoirs to said array of separation channels, wherein at least one anode reservoir is connected to two or more separation channels of said array of separation channels; and connecting an array of waste reservoirs to said array of separation channels, wherein at least one waste reservoir of said array of waste reservoirs is connected to two or more sample reservoirs of said array of sample reservoirs.
 35. The method of claim 34, wherein a distance from each cathode reservoir to a point where a sample reservoir of said array of sample reservoirs is connected to said channel is approximately equal for each separation channel.
 36. The method of claim 34, wherein a distance from each anode reservoir to a point where a sample reservoir of said array of sample reservoirs is connected to said channel is approximately equal for each separation channel.
 37. A capillary array electrophoresis plate, comprising:a first set of cathode reservoirs positioned near an outer perimeter of the plate; a second set of anode reservoirs positioned near a center of the plate, wherein the number of reservoirs in said second set is fewer than the number of reservoirs in said first set; and an array of separation channels connecting the first set of reservoirs to the second set of reservoirs.
 38. The capillary array electrophoresis plate of claim 37, wherein said array of separation channels radially connect said first set of reservoirs to said second set of reservoirs.
 39. A capillary array electrophoresis plate, comprising:a separation channel formed in said plate connected to a cathode reservoir and an anode reservoir; a first cross channel connected to the separation channel at a first location, wherein a first end of the first cross channel connects to a first waste reservoir and a second end connects to a first sample reservoir; and a second cross channel connected to the separation channel at a second location; wherein a first end of the second cross channel connects to the first waste reservoir and a second end connects to a second sample reservoir.
 40. A capillary array electrophoresis plate comprising:an array of microfabricated separation channels formed on a surface of a first microfabricated substrate and a corresponding surface of a second substrate bonded to the surface of said first substrate, each of said channels having a first end and a second end; an array of sample reservoirs connected to said array of separation channels wherein each of a plurality of individual separation channels are each in fluid communication with at least one dedicated sample reservoir; and an array of waste reservoirs connected to said separation channels, wherein at least one waste reservoir is multiplexed to two or more of said channels, wherein the capillary array electrophoresis plate has H holes, and wherein H is approximately equal to 5N/4, with N being the number of samples to be processed.
 41. A capillary array electrophoresis plate comprising:a microfabricated separation channel formed on a surface of a first microfabricated substrate and a corresponding surface of a second substrate bonded to the surface of said first substrate, said channel being connected to a cathode reservoir on one end and an anode reservoir on the other end; and two or more sample reservoirs sharing a waste reservoir and connected to the separation channel, wherein the capillary array electrophoresis plate has H holes, and wherein H is approximately equal to 5N/4, with N being the number of samples to be processed. 